Effect of sex and milk replacer with or without supplemental carnitine and arginine on growth characteristics, carcass, and meat quality of artificially reared low-birth weight pigs.

This study compared milk replacer either remaining unsupplemented (CON) or supplemented with 0.5 g L-carnitine plus 16.7 g L-arginine/kg (CarArg) and fed to 48 low-birth weight (L-BtW) artificially reared piglets (24 per group) from days 7 to 28 of age. Eight farrowing series were needed to complete the study. On day 28, the lightest piglets were slaughtered, and the heaviest pigs were weaned. The heaviest pigs were weaned on day 28 and offered free access to a starter (weaning to 25 kg body weight [BW]), grower (25 to 60 kg BW), and finisher diet (60 to 96 kg BW on day 170 of age). After euthanization on days 28 and 170, blood was sampled for assessment of serum metabolite and hormone concentrations, and the semitendinosus muscle (STM) was weighed, and later subjected to enzyme activity analysis and assessment of myofiber characteristics. In the 170-d-old pigs carcass and meat quality traits were assessed. Growth data were analyzed accordingtoatwo-way analysis of variance (ANOVA), with dietary treatment and farrowing series as fixed effects, while remaining data were analyzed with dietary treatment, sex, their interaction, and farrowing series as main factors. Dietary treatments affected (P ≤ 0.049) muscle enzyme activity at both day 28, with greater citrate synthase (CS) and LDH activities and lower HAD:CS ratio in STM light portion, and lower LDH:CS ratio in STM dark portion, and 170 of age with lower HAD:CS ratio. In the starter period, CarArg pigs had greater average daily gain (P = 0.021) and average daily feed intake (P = 0.010). At slaughter, these pigs had lower (P = 0.013) glucose and greater (P = 0.022) urea serum concentrations. However, supplementing the milk replacer with carnitine and arginine had no long-term effects on growth performance, carcass composition, and meat quality of L-BtW pigs. In addition, muscle morphology and myofiber-related properties remained unaffected by the supplementation.

Breeding efforts to increase litter size in modern sows have inadvertently reduced the average birth weight of piglets, resulting in a higher number of piglets born with low-birth weight. These piglets are indeed vulnerable from birth and display relatively poor growth potential from a very early stage. For this reason, artificial rearing strategies are potentially a management option to improve the growth of these runt piglets. With an artificial rearing system, it is possible to provide specialized diets already during the suckling period, with inclusion of specific nutrients in certain concentrations suggested to improve the growth of runt piglets. Using an artificial rearing system allows for the provision of specialized diets during the suckling phase, which includes specific nutrients aimed at enhancing the growth of underdeveloped piglets. However, in the current experiment, the particular nutrients and their dosages did not significantly improve growth or other characteristics compared to the control group.

Sustainable pig diets: partial grain replacement with former food products and its impact on meat quality.

This study investigated the effects of salty and sugary former foodstuff products (FFPs) on the quality traits and meat composition of 36 male castrated pigs (Swiss Large White breed) as well as sensory characteristics of the loins. The animals were fed three different diets for both the growing (G) and finishing (F) phases: (1) a standard diet (ST), 0% FFPs; (2) a diet with 30% of sugary FFPs (e.g., chocolate, biscuits, cakes) as a replacement for traditional ingredients (SU); and (3) a diet with 30% of salty FFPs (e.g., bread, pasta, and breadsticks) as a replacement for traditional ingredients (SA). For a comprehensive assessment of meat quality, protein and fat content in the LD were analyzed. AA and FA profile were determined both in the LD and backfat. Meat quality traits such as pH and temperature, thawing, cooking and drip losses, and shear force have been evaluated. Then, pork loins have been assessed for sensory attributes by a trained sensory panel. The SA diet decreased 20:5 n-3 levels (P < 0.001) in the muscle and 22:5 n-3 levels (P < 0.05) in both muscle and backfat but increased (P < 0.05) the ratio of mono-unsaturated to saturated fatty acids compared to the ST group. Both the SU and SA diets elevated (P < 0.001) the n-6:n-3 fatty acids ratio compared to the ST diet. Dietary treatments did not affect other meat quality traits. Regarding sensory attributes, the loin from pigs fed with SU and SA diets were sweeter (P < 0.001). Loins of SA pigs were more tender (P < 0.001), had a more intense pork aroma (P < 0.001) and had more flavor (P < 0.01) compared to ST loins. Overall, the use of FFPs affected the fatty acid profile of pork while improving the sensory quality of the loins, with no negative effects observed on the technological and nutritional quality of the meat.

Pigs are ideal species to convert food losses, also named former food products (FFPs), into animal proteins. The present study investigated the impact of incorporating sugary and salty FFPs into the diets of growing and finishing pigs on meat quality and its sensory characteristics. Our study showed that, while technological meat quality remained largely unaffected, the dietary treatments led to slight alterations of meat and backfat fatty acids profile. Moreover, a panel test for sensory analyses revealed that loins from both the sugary and salty ingredients-fed pigs were perceived as sweeter, and loins from pigs fed salty ingredients were noted for increased tenderness, intense pork aroma, and flavor. Overall, FFPs inclusion into pig diets had no detrimental effects on technological or nutritional aspects of the meat, confirming their potential use as alternative animal feed.

Lupinus albus γ-Conglutin: New Findings about Its Action at the Intestinal Barrier and a Critical Analysis of the State of the Art on Its Postprandial Glycaemic Regulating Activity.

γ-Conglutin (γ-C) is the glycoprotein from the edible seed L. albus, studied for long time for its postprandial glycaemic regulating action. It still lacks clear information on what could happen at the meeting point between the protein and the organism: the intestinal barrier. We compared an in vitro system involving Caco-2 and IPEC-J2 cells with an ex vivo system using pig ileum and jejunum segments to study γ-C transport from the apical to the basolateral compartment, and its effects on the D-glucose uptake and glucose transporters protein expression. Finally, we studied its potential in modulating glucose metabolism by assessing the possible inhibition of α-amylase and α-glucosidase. RP-HPLC analyses showed that γ-C may be transported to the basolateral side in the in vitro system but not in the pig intestines. γ-C was also able to promote a decrease in glucose uptake in both cells and jejunum independently from the expression of the SGLT1 and GLUT2 transporters.

Accumulation and decontamination kinetics of PCBs and PCDD/Fs from grass silage and soil in a transgenerational cow-calf setting.

Polychlorinated biphenyls (PCBs) and dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) are bioaccumulative pollutants that endanger bovine food safety. Bioaccumulation depends, among others, on the physiological dynamics of the cow's reproductive cycle. However, recent studies have focused only on near steady-state situations. Thus, the effects of animal physiology on PCB + PCDD/F transfer from grass silage and soil to cows' blood, adipose tissue, and milk and subsequently to suckling calves during gestation and lactation were investigated. In the exposed group, nine cows ate a grass silage/contaminated soil mixture (6.6 ± 0.8 µg iPCBs and 2.6 ± 0.4 ng dlPCB + PCDD/F TEQ kgDM-1) for 109 days prepartum until 288 days in milk (DIM). Four of these cows underwent decontamination after DIM164, receiving the same clean grass silage as the four control cows during the experiment. Calves were fed the milk of their respective mothers. In the exposed group, transgenerational bioaccumulation occurred until DIM164, with calf blood and adipose tissue PCB + PCDD/F concentrations reaching levels twice as high as those in their respective mothers. Transfer rates from oral intake to milk ranged from 0.1 up to 42%, depending on pollutant congener, dietary treatment, and reproductive parity of the cow. Congener and parity also influenced the decontamination half-lives of milk. In decontaminated calves, declines in adipose tissue PCB + PCDD/F concentrations coincided with increases in body fat mass. Therefore, it is essential to know the physiological characteristics of cattle, exposure dose and duration, and physicochemical compound properties to perform reliable transfer assessments.

Gallic acid affects intestinal-epithelial-cell integrity and selected amino-acid uptake in porcine in vitro and ex vivo permeability models.

Gallic acid (GA) is widely used as a dietary supplement due to several health-promoting effects, although its effects on intestinal-epithelial-cell integrity and transport remain mostly unknown. The present study aims to clarify the effects of GA on tight junctions and intestinal nutrient uptake through in vitro and ex vivo models. Both intestinal porcine enterocyte cell line-J2 cells and porcine middle-jejunum segments were treated with 5 (T5), 25 (T25) and 50 (T50) µm GA and mounted in Ussing chambers to determine transepithelial resistance (TEER), claudin-1 (CLDN1), occludin (OCLN), zonula occludens-1 (ZO-1) protein (in tissues and cells) and mRNA (in cells) expression. In addition, uptake of l-glutamate (l-Glut), l-arginine (l-Arg), l-lysine (l-Lys) and l-methionine (l-Meth) together with cationic-amino-acid transporter-1 (CAT-1) and excitatory-amino-acid transporter-3 (EAAT3) expression was evaluated. No apoptosis was observed in GA-treated cells, but TEER and CLDN1 protein abundance was lower with T50 compared with untreated cells. l-Arg and l-Lys uptake was greater with T5 than with T25 and T50. Ex vivo, T50 decreased the TEER values and the protein levels of CLDN1, OCLN and ZO-1, whereas T5 and T25 only decreased CLDN1 protein expression compared with untreated tissues. Moreover, T25 increased l-Glut and l-Arg uptake, the latter confirmed by an increased protein expression of CAT-1. GA influences intestinal uptake of the tested cationic amino acids at low concentrations and decreases the intestinal-cell barrier function at high concentrations. Similarities were observed between in vitro and ex vivo, but different treatment times and structures must be considered.

Effect of Supplementing Hydrolysable Tannins to a Grower-Finisher Diet Containing Divergent PUFA Levels on Growth Performance, Boar Taint Levels in Back Fat and Intestinal Microbiota of Entire Males.

A retrospective data analysis suggested that the levels of boar taint compounds depend on the polyunsaturated fatty acid (PUFA) level of the adipose tissue (AT) being significantly greater in the unsaturated AT. In addition, we recently reported that hydrolysable tannins (HTs) offered to entire males (EMs) reduce skatole and, to a greater extent, indole levels in the AT. Thus, the objective of the study was to determine the impact of HTs and a high dietary level of PUFA on growth performance and board taint compounds in EMs. In addition, the interaction between PUFA and HTs on gut microbiota and its link to intestinal skatole and indole production was investigated. At 25 kg BW, 44 EM originating from 11 litters were randomly assigned within litter to four dietary treatments. Two basal grower (25-60 kg BW) and finisher (60-105 kg BW) diets containing either 2% soy oil (H = high PUFA level) or 2% tallow (L = low PUFA level) were formulated. The H and L diets were either supplemented (H+/L+) or not (H-/L-) with 3% chestnut extract containing 50% HTs. The pigs had ad libitum access to the diets and were slaughtered at 170 days of age. The microbiota composition was investigated through the 16S rRNA gene sequences obtained by next-generation sequencing (Illumia MiSeq platform, San Diego, CA, USA) and analyzed with a specific packages in R, version 3.5.0. Regardless of the PUFA content, the EMs fed the H+ diets were 2% (p < 0.01) less feed efficient overall. This was due to the slower (p = 0.01) growth in the finisher period despite similar feed intake. Carcass characteristics were not affected by the diets. Regardless of HT feeding, the PUFA level in the AT of the H pigs was 10% greater (p = 0.05) than in the L pigs. The indole level tended (p = 0.08) to be 50% lower in the H+ group. Surprisingly, the pigs that were fed diet H- had greater skatole levels than those fed diet L-, with intermediate skatole levels in the H+ and L+. Independent of the PUFA level, the HTs decreased bacteria abundance and qualitatively affected the microbiota composition. In conclusion, these data do not confirm that boar taint compound levels were related to PUFA levels in the AT. However, HTs can be considered to be a promising alternative to conventional antibacterial additives, with no detrimental effects on pig gut health and with appealing properties for reducing the synthesis of the main components of boar taint.

Punicalagin increases glutamate absorption in differentiated Caco-2 cells by a mechanism involving gene expression regulation of an EAAT3 transporter.

This study investigates, for the first time, the ability of punicalagin to modulate intestinal glutamate uptake by upregulation of the expression of one of its transporters present on the enterocyte membrane. The use of an Ussing chamber revealed an increase in glutamate transport in differentiated Caco-2 cells after punicalagin treatment for 24 h. This cell line constitutively expresses two glutamate transporters: EAAT1 and EAAT3. In response to punicalagin, the expression of EAAT3 was increased, at both mRNA and protein levels, but not that of EAAT1. Transfection with EAAT3-targeting siRNA specifically altered basal and induced EAAT3 gene expression, decreasing the positive effect of punicalagin on glutamate uptake. These data confirmed the involvement of EAAT3 in increasing glutamate uptake by enterocytes after punicalagin treatment.

An improved DNA method to unambiguously detect small hive beetle Aethina tumida, an invasive pest of honeybee colonies.

The scavenger and invasive species Aethina tumida threatening the honey bee has been recently introduced in Europe. We present a new, reliable and rapid multiplex real-time PCR for efficient diagnostics enabling surveillance programs. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of nursing or mentoring by adult cows on physical activity, performance and meat quality of fattening beef calves kept on alpine pastures.

BACKGROUND: Compared with weaned calves, nursed calves benefit from access to milk as nutrient-dense feed and mentoring by their dams. Both may be decisive for growth under difficult conditions such as grazing on alpine pastures, where experience could play an essential role. These factors were investigated separately by comparing nursed calves with calves weaned at 3 months of age and grazing with or without a mentor cow (n = 8 calves). RESULTS: Weaned calves with mentor cows spent less time lying and walked more than nursed calves. Irrespective of whether they had a mentor cow or not, the weaned calves grew slower than the nursed calves (357, 428 and 1324 g day-1 respectively). This also adversely affected slaughter weight, dressing percentage, conformation and fat cover at slaughter. In general, mentoring had no effect. Meat quality did not differ whether weaned calves were mentored or not. Nursed calves were superior in intramuscular fat content, tenderness and water-holding capacity. CONCLUSION: The availability of milk appeared to be much more important to the calves under demanding grazing conditions than did mentoring. The differences in growth rate were accompanied by unexpectedly large differences in meat quality, showing that early weaning was not suitable for this system.© 2016 Society of Chemical Industry.

Forage legumes rich in condensed tannins may increase n-3 fatty acid levels and sensory quality of lamb meat.

BACKGROUND: Tannins intensively interact with rumen microbes, which is expected to have consequences for meat quality. RESULTS: Silages prepared from birdsfoot trefoil (BT), sainfoin (SF), alfalfa (AF) or red clover (RC) were fed alone to 48 lambs. The SF contained five times more condensed tannins than BT, the other tanniferous plant. Growth and carcass performance, but not general meat quality, was reduced with BT and SF compared to AF and RC. Lambs fed SF had half the skatole levels in the perirenal fat than AF-fed lambs. The longissimus thoracis et lumborum muscle of the SF-fed lambs, compared to RC and BT, had a lower intensity for 'livery' and 'sheepy' flavors but a stronger 'grassy' flavor. The intramuscular fat of BT- and SF-fed lambs contained less saturated and more polyunsaturated fatty acids, especially 20:4n-6, 20:3n-6, 20:5n-3 and 22:5n-3, with SF being more efficient than BT. CONCLUSION: The SF was most promising to increase beneficial fatty acids and to reduce skatole content in lamb meat.

Image thresholding techniques for localization of sub-resolution fluorescent biomarkers.

In this article, we explore adaptive global and local segmentation techniques for a lab-on-chip nutrition monitoring system (NutriChip). The experimental setup consists of Caco-2 intestinal cells that can be artificially stimulated to trigger an immune response. The eventual response is optically monitored using immunofluoresence techniques targeting toll-like receptor 2 (TLR2). Two problems of interest need to be addressed by means of image processing. First, a new cell sample must be properly classified as stimulated or not. Second, the location of the stained TLR2 must be recovered in case the sample has been stimulated. The algorithmic approach to solving these problems is based on the ability of a segmentation technique to properly segment fluorescent spots. The sample classification is based on the amount and intensity of the segmented pixels, while the various segmenting blobs provide an approximate localization of TLR2. A novel local thresholding algorithm and three well-known spot segmentation techniques are compared in this study. Quantitative assessment of these techniques based on real and synthesized data demonstrates the improved segmentation capabilities of the proposed algorithm.

NutriChip: nutrition analysis meets microfluidics.

This focus article introduces the concept of NutriChip, an integrated microfluidic platform for investigating the potential of the immuno-modulatory function of dairy food. The core component of the NutriChip is a miniaturized artificial human gastrointestinal tract (GIT), which consists of a confluent layer of epithelial cells separated from a co-culture of immune cells by a permeable membrane. This setting creates conditions mimicking the human GIT and allows studying processes that characterize the passage of nutrients though the human GIT, including the activation of immune cells in response to the transfer of nutrients across the epithelial layer. The NutriChip project started by developing a biologically active in vitro cellular system in a commercial Transwell co-culture system. This Transwell system serves as a reference for the micro-scale device which is being developed. The microfluidic setup of NutriChip allows monitoring of the response of immune cells to pro-inflammatory stimuli, such as lipid polysaccharide (LPS), and to the application of potentially anti-inflammatory dairy food. This differential response will be quantified by measuring the variation in expression of pro-inflammatory cytokines, including interleukin 1 (IL-1) and interleukin 6 (IL-6), secreted by the immune cells, and this is achieved by using a dedicated optical imager. A series of dairy products will be screened for their anti-inflammatory properties using the NutriChip system and, finally, the outcome of the NutriChip will be validated by a human nutrition trial. Therefore, the NutriChip platform offers a new option to evaluate the influence of food quality on health, by monitoring the expression of relevant immune cell biomarkers.

The NutriChip project--translating technology into nutritional knowledge.

Advances in food transformation have dramatically increased the diversity of products on the market and, consequently, exposed consumers to a complex spectrum of bioactive nutrients whose potential risks and benefits have mostly not been confidently demonstrated. Therefore, tools are needed to efficiently screen products for selected physiological properties before they enter the market. NutriChip is an interdisciplinary modular project funded by the Swiss programme Nano-Tera, which groups scientists from several areas of research with the aim of developing analytical strategies that will enable functional screening of foods. The project focuses on postprandial inflammatory stress, which potentially contributes to the development of chronic inflammatory diseases. The first module of the NutriChip project is composed of three in vitro biochemical steps that mimic the digestion process, intestinal absorption, and subsequent modulation of immune cells by the bioavailable nutrients. The second module is a miniaturised form of the first module (gut-on-a-chip) that integrates a microfluidic-based cell co-culture system and super-resolution imaging technologies to provide a physiologically relevant fluid flow environment and allows sensitive real-time analysis of the products screened in vitro. The third module aims at validating the in vitro screening model by assessing the nutritional properties of selected food products in humans. Because of the immunomodulatory properties of milk as well as its amenability to technological transformation, dairy products have been selected as model foods. The NutriChip project reflects the opening of food and nutrition sciences to state-of-the-art technologies, a key step in the translation of transdisciplinary knowledge into nutritional advice.

The effects of early post-mortem pH and ultimate pH on level and amount of destructured zones in cooked cured hams.

Effects of early (1h p.m. and 3h p.m.) and ultimate pH (24h p.m.) on level and amount of destructured zones in cooked cured hams were evaluated. In experiment 1, electrically stimulated (50 V, 14 Hz, 2 x 90s) and non-stimulated carcass halves, both in combination with two cooling procedures (2 degrees C from 30 min p.m. vs. 120 min p.m.) resulted in 1.5-35.2g/kg destructured zones in silversides and 58.4-120.0 g/kg destructured zones in topsides. A high temperature 1h p.m. in silversides (P=0.067) and topsides (P=0.054) was identified as the most important predictor for the defect. In experiment 2, cooked cured hams from topsides selected according to ultimate pH groups (pH<5.5, pH 5.5-5.7, pH>5.7) showed between 12.3 and 61.8 g/kg destructured zones. Ultimate pH was specified as most important, however, statistically still not significant (P=0.135) predictor for the defect. Chemical analysis resulted in low crude ash and high dry matter content as being characteristic for the defect.

Regulation of arginase pathway in response to wall shear stress.

OBJECTIVE: Alterations of wall shear stress can predispose the endothelium to the development of atherosclerotic plaques. Ample evidence indicates that arginase expression and/or activity correlates with several risk factors for cardiovascular disease including atherosclerosis. We investigated the regulation of arginase pathway in response to distinct patterns of wall shear stress. METHODS: Isolated porcine endothelial cells and carotid arterial segments were perfused under unidirectional high shear stress (HSS) or oscillatory shear stress (OSS) for 1 and 3 days. Arginase I and II expression, cellular localization and enzyme activity were, respectively, assessed by Western blot, immunohistochemistry and colorimetric determination of urea. The contribution of arginase to the processes of endothelial dysfunction, cell proliferation and arterial remodeling induced by OSS was evaluated by administration of the arginase inhibitor N-omega-hydroxy-nor-l-arginine (nor-Noha). RESULTS: Only arginase II isoform was detected on porcine carotid endothelial cells and on carotid artery. Exposure of arteries to OSS increased arginase II expression and activity as compared to HSS. Inhibition of arginase by nor-Noha improved NO-dependent endothelial function and decreased total vascular ROS formation in arteries submitted to OSS. In addition, inhibition of arginase activity decreased smooth muscle cell proliferation rate with no effect on collagen content after OSS. CONCLUSIONS: Exposure of carotid artery to oscillatory flow induced a more pronounced activation of arginase as compared to HSS. Inhibition of arginase in arteries exposed to OSS improved NO-dependent endothelial function and decrease smooth muscle cell proliferation rate, both processes are important for the focal development of atherosclerotic plaque.

Reduced cyclic stretch, endothelial dysfunction, and oxidative stress: an ex vivo model.

BACKGROUND: The objective of this study was to investigate whether reduction of cyclic circumferential stretch will impair endothelial function and elevate basal levels of oxidative stress, both known risk factors linked to cardiovascular disease. METHODS: Ex vivo and in vitro models were used to perfuse porcine carotid arteries and porcine endothelial cells, respectively, for 24 h. In both cases, one group was allowed to stretch naturally when exposed to a pulse shear stress (6+/-3 dynes/cm(2)) combined with a pulse pressure of 80+/-10 mmHg, yielding a physiological cyclic stretch of 4-5%. This group was compared to a reduced stretch group, achieved by wrapping the arterial segment with a silicon band or by seeding the endothelial cells inside less compliant tubes, decreasing cyclic stretch to 1%. RESULTS: The experimentally reduced compliance caused a significant decrease in bradykinin-dependent vascular relaxation. Reduced compliance significantly decreased the phosphorylation of serine 1177 (Ser1177) on eNOS, suggesting the activity of eNOS was decreased. Overall production of reactive oxygen species was increased by reducing compliance, as visualized with DHE. Finally, p22-phox and p47-phox, key players in the superoxide-generating NAD(P)H oxidase, were also up-regulated by reduced compliance. CONCLUSIONS: These findings point out how reduced arterial compliance increases the risk of arterial disease by creating a less functional endothelium, interrupting the eNOS activation pathway, and increasing the vascular levels of oxidative stress.

Autonomous effects of shear stress and cyclic circumferential stretch regarding endothelial dysfunction and oxidative stress: an ex vivo arterial model.

Cyclic circumferential stretch and shear stress caused by pulsatile blood flow work in concert, yet are very different stimuli capable of independently mediating endothelial function by modulating eNOS expression, oxidative stress (via production of superoxide anion) and NO bioavailability. Porcine carotid arteries were perfused using an ex vivo arterial support system for 72 h. Groups we created by combining normal (5%) and reduced (1%) stretch with high shear (6 +/- 3 dynes/cm(2)) and oscillatory shear (0.3 +/- 3 dynes/cm(2)) stress while maintaining a pulse pressure of 80 +/- 10 mm Hg. Oscillatory flow and reduced stretch both proved detrimental to endothelial function, whereas oscillatory flow alone dominated total endogenous vascular wall superoxide anion production. Yet, when superoxide anion production was analyzed in just the endothelial region, we observed that it was modulated more significantly by reduced cyclic stretch than by oscillatory shear, emphasizing an important distinction between shear- and stretch-mediated effects to the vascular wall. Western blotting analysis of eNOS and nitrotyrosine proved that they too are more significantly negatively modulated by oscillatory flow than by reduced stretch. These findings point out how shear and stretch stimulate regions of the vascular wall differently, affecting NO bioavailability and contributing to vascular disease.

Effects of reduced cyclic stretch on vascular smooth muscle cell function of pig carotids perfused ex vivo.

BACKGROUND: With advancing age arteries stiffen, reducing arterial compliance and leading to the development of systolic hypertension and to a substantial increase in pulse pressure. An augmented pulse pressure can be a predictor of the development of hypertension, which has been linked to several cardiovascular diseases including atherosclerosis, and to pathologies such as diabetes and renal dysfunction. In this study, we tested the hypothesis that reduced wall compliance induces pulse-pressure-mediated changes in arterial wall metabolism and remodeling. METHODS: Porcine carotid arteries were perfused for 24 h using an ex vivo arterial support system. Control arteries were exposed to a pulse shear stress (6 +/- 3 dynes/cm(2)) combined with a pulse pressure of 80 +/- 10 mm Hg, yielding a physiological cyclic stretch of 4-5%. A reduced compliance group was also studied, in which arteries were wrapped with an external band, thereby decreasing cyclic stretch to levels <1%. RESULTS: The experimentally reduced compliance caused a decreased contraction capacity induced by norepinephrine(NE), and this was associated with lower levels of alpha-smooth muscle cell-actin (alpha-SMC-actin) and desmin protein expressions. Arteries that were exposed to a reduced cyclic stretch exhibited a higher level of matrix metalloproteinase-2 (MMP-2) expression activity as well as an increase in Ki67 expression, thereby suggesting that matrix degradation and cellular proliferation had been initiated. Furthermore, the expression of plasminogen activator inhibitor-1 (PAI-1) in stiffened arteries was lower than in the control arteries. CONCLUSIONS: These findings underline the importance of cyclic stretch in the maintenance of a differentiated and fully functional phenotype of vascular SMCs, as well as in the regulation of migratory properties, proliferation, and matrix turnover.

Transcriptional and post-transcriptional regulation of preproendothelin-1 by plaque-prone hemodynamics.

OBJECTIVE: Plaque-prone areas are exposed to a particular hemodynamic environment characterized by a low mean shear stress value and a cyclic reversal flow. This mechanical environment, also termed oscillatory shear stress (OSS), induces the expression of several pro-atherogenic genes in the endothelial cells including the preproendothelin-1 (ppET-1) gene. The present paper investigates the molecular mechanisms of this induction. METHODS AND RESULTS: Several deletional mutants of ppET-1 gene promoter were cloned upstream of a luciferase gene and transiently transfected in bovine arterial endothelial cells that were further exposed to plaque-prone hemodynamics. After 24h of flow exposure, analysis of the transfected cells showed that a proximal promoter of 156 base pairs length retained OSS responsiveness. Mutation of an activator protein-1 (AP-1) binding site present in this minimal promoter completely abolished its activation by OSS. Consistently, electrophoresis mobility shift assay revealed a sustained activation of AP-1 transcription factor in endothelial cells exposed to OSS. In addition to the transcriptional activation, we demonstrated that OSS also induces a stabilization of ppET-1mRNA through the 3'-untranslated region (3'-UTR) of this gene. Fluvastatin, a drug known to improve endothelial function, was shown to prevent OSS up-regulation of the ppET-1 gene expression. Under this flow condition, fluvastatin affects ppET-1 gene expression via inhibition of its promoter activity without affecting ppET-1mRNA stability. CONCLUSIONS: The present study demonstrate that plaque-prone hemodynamic induces ppET-1 gene expression by both transcriptional and post-transcriptional mechanisms via an activation of AP-1 transcriptional factor and stabilization of mRNA. The transcriptional up-regulation of ppET-1 was shown to be fluvastatin sensitive.

RGD-Functionalized polymer brushes as substrates for the integrin specific adhesion of human umbilical vein endothelial cells.

This report demonstrates the feasibility of surface-initiated atom transfer radical polymerization to prepare thin polymer layers ("brushes") that can be functionalized with short peptide ligands and which may be of use as coatings to promote endothelialization of blood-contacting biomaterials. The brushes are composed of poly(2-hydroxyethyl methacrylate) (PHEMA) or poly(poly(ethylene glycol) methacrylate) (PPEGMA), which do not only suppress non-specific adhesion of proteins and cells but also contain hydroxyl groups that can be used to introduce small peptide ligands. A protocol has been developed that allows functionalization of the brushes with RGD containing peptide ligands resulting in surface concentrations ranging from approximately 0.5-12 pmol/cm(2). At peptide surface concentrations >1-5.3 pmol/cm(2), human umbilical vascular endothelial cells (HUVECs) were found to adhere and spread rapidly. A difference in size and morphology of focal adhesions between HUVECs immobilized on PHEMA and PPEGMA brushes was observed. It is proposed that this is due to the increased ethylene glycol spacer length and hydrophilicity of the PPEGMA brushes, which may lead to increased ligand mobility and reduced ligand-integrin affinity. HUVECs immobilized on the polymer brushes were also found to be able to retain homeostasis when exposed to shear stresses that simulated arterial blood flow.